TY  - JOUR
AU  - Schneider, K.
AU  - Dimroth, B. M.
AU  - Bott, M.
TI  - Biosynthesis of the prosthetic group of citrate lyase
JO  - Biochemistry
VL  - 39
SN  - 0006-2960
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - PreJuSER-26938
SP  - 9438 - 9450
PY  - 2000
N1  - Record converted from VDB: 12.11.2012
AB  - Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.
KW  - Acyl Carrier Protein: biosynthesis
KW  - Amino Acid Sequence
KW  - Apoproteins: biosynthesis
KW  - Carbon-Sulfur Ligases: biosynthesis
KW  - Carbon-Sulfur Ligases: genetics
KW  - Coenzyme A: biosynthesis
KW  - Enzyme Precursors: metabolism
KW  - Escherichia coli: enzymology
KW  - Escherichia coli: genetics
KW  - Genes, Bacterial
KW  - Molecular Sequence Data
KW  - Multienzyme Complexes: biosynthesis
KW  - Multienzyme Complexes: chemistry
KW  - Multienzyme Complexes: genetics
KW  - Multigene Family
KW  - Operon
KW  - Oxo-Acid-Lyases: biosynthesis
KW  - Oxo-Acid-Lyases: chemistry
KW  - Oxo-Acid-Lyases: genetics
KW  - Structure-Activity Relationship
KW  - Acyl Carrier Protein (NLM Chemicals)
KW  - Apoproteins (NLM Chemicals)
KW  - Enzyme Precursors (NLM Chemicals)
KW  - Multienzyme Complexes (NLM Chemicals)
KW  - dephosphocoenzyme A (NLM Chemicals)
KW  - Coenzyme A (NLM Chemicals)
KW  - Oxo-Acid-Lyases (NLM Chemicals)
KW  - citrate (pro-3S)-lyase (NLM Chemicals)
KW  - Carbon-Sulfur Ligases (NLM Chemicals)
KW  - long-chain-fatty-acid-(acyl-carrier-protein) ligase (NLM Chemicals)
KW  - citrate (pro-3S)-lyase ligase (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:10924139
UR  - <Go to ISI:>//WOS:000088593300042
UR  - https://juser.fz-juelich.de/record/26938
ER  -